**4.** *Campylobacter*

In contrast, the proportions of FQ resistance in humans were significantly lower (11%) [5] and has remained at a relatively stable level since 2009 [31]. The biggest share of FQ resistance was detected in Northern Europe, mostly on the account of Estonia (36.0%), Finland (26.3%), Norway (24.7%) and Ireland (22.9%) (**Figure 4**). Human-associated serovars commonly detected in Europe [32–35] that frequently exhibited FQ resistance were *S.* Infantis (23.4%)

FQ resistance is a result of a complex mechanism, but it is still not fully understood [36]. Many point mutations in the genes encoding for gyrase and topoisomerase, the two enzymes that are inhibited by FQ, were identified as the causative agents [37]. In addition, plasmids may harbour genes for efflux pumps, target protection proteins or drug-modifying enzymes [38]. Resistance to third-generation cephalosporins was rare in humans as well as in livestock [5], yet when combined with FQ resistance, it poses a serious risk to human health, in terms of reducing the efficiency of these drugs against salmonellosis and thus leaving only the reserve antimicrobials as a feasible therapy option [24]. Resistance to cephalosporins is conferred by genes encoding for AmpC β-lactamase as well as for various extended spectrum β-lactamase (ESBL) that can be located on plasmids. Such isolates were observed in Germany [39] and are assumed to have clonally spread from livestock to humans. Worryingly, in Portugal, more than a third of the isolates from broiler meat (39.4%) exhibited resistance to cefotaxime and ceftazidime and in Italy 12% from broilers [5]. In addition, combined resistance to FQ and cephalosporins was detected in poultry and humans in Spain, Belgium and France [40, 41]. *Salmonella* in the food production chain presents an important reservoir of genetic resistance determinants, which could be mobilised and transferred *via* the food chain to either other human pathogens or commensal bacteria [42]. Notably, importation of meat products [43] and travelling in endemic areas [44], where the rates of resistance to critically important anti-

microbials are alarmingly high [45], were linked to the global spread of MDR strains.

In general, 26.5% of the human isolates of *Salmonella* and 50.3% of broiler meat displayed MDR phenotype (defined as resistant to at least three antimicrobials of the nine antimicrobial classes tested). The highest prevalence of MDR isolates from humans was observed in Portugal (51%) and from broiler meat in Slovenia (100%) [5]. MDR strains isolated from pigs in Germany were associated with integrons, which might have an important role in dissemi-

The majority of MDR isolates belonged to serovars *S.* Infantis and *S.* Kentucky. Among human isolates of *S.* Kentucky, which is the seventh most common serovar, MDR was recorded at extremely high levels (76.3%) [5]. *S.* Kentucky ST198 clone that is displaying high-level resistance to ciprofloxacin and frequently also to amoxicillin, streptomycin, spectinomycin, gentamicin, sulfamethoxazole and tetracycline has been imported from North Africa and has been widely spread across Europe in humans and food production chain [32]. In addition, acquisition of extended-spectrum β-lactamase, plasmid-encoded cephalosporinase or carbapenemase in this clone was detected in Mediterranean area [47] and in Poland [33]. Combined resistance was also detected in *S.* Kentucky from humans and livestock in Belgium, Luxembourg, Malta, the Netherlands and Germany [5].

**3.4. MDR and combined resistance to fluoroquinolones and third-generation** 

and *S.* Kentucky (85.8%) [5].

18 Antimicrobial Resistance - A Global Threat

**cephalosporins**

nation of resistance [46].

#### **4.1. Prevalence of** *Campylobacter* **in the food chain**

Whilst *Campylobacter,* with 246,307 confirmed infections in 2016 and 6.1% increase relative to 2015, accounted for the majority of zoonoses in Europe, the death toll was low (0.03%). The highest notification rate per 100,000 population was observed in Eastern Europe (71.4), followed by Western (65.7), Southern (56.3) and Northern Europe (55.0). Czech Republic (228.2) and Slovakia (140.5) were the countries with the highest prevalence [7].

Campylobacter*s* were most frequently detected in turkeys (65.3%) and meat thereof (11%) as well as in broilers (27.3%) and meat thereof (36.7%) [7], making the poultry food production chain the main source of contamination. This is in concordance to data from several reports [50, 51]. The prevalence in retail poultry meat was, however, reported even up to almost 90% [50]. *Campylobacter* was also detected in cattle [52], pigs [53] and sheep [54]. Contaminated farm environment or equipment as well as the presence of *Campylobacter* in other animals and wildlife, were significantly associated with the prevalence of *Campylobacter* in poultry [55]. Furthermore, recent data suggest that human clinical *C. jejuni* isolates in Central Europe can be attributed to domesticated poultry, cattle livestock and environmental sources [56].

Outbreaks can be traced back to several sources (e.g., raw milk [57], water [58] and chicken liver pate [59]) and even associated with antimicrobial-resistant strains [57]. However, a limited number of highly contaminated products are most probably responsible for the majority of *Campylobacter* infections. Effective and harmonised surveillance systems, especially in the poultry food production chain, that are oriented towards categorising risks should thus be established [50].

#### **4.2. Antimicrobial resistance in** *Campylobacter*

*Campylobacter* spp. in 2016 displayed extremely high resistance levels to FQ, which is particularly worrisome, as FQ are used as the first-line drugs against campylobacteriosis. Consequently, in some EU countries, FQ therapy of campylobacteriosis is no longer feasible. In average, the highest share of resistant isolates was detected in poultry and meat thereof, especially in the Member States of Southern and Eastern Europe (**Figure 5**) [5]. Data suggest that the use of FQ in livestock, specifically pigs, selects for FQ-resistant strains and accelerates the dissemination of such strains [60].

**4.3. FQ resistance and combined/MDR resistance in** *Campylobacter*

rates of FQ resistance in endemic areas [72] are therefore of concern.

sources of resistance, closely followed by broilers and meat thereof [5].

Germany, Austria) [74].

to FQ and erythromycin in *C. coli* from humans [5].

FQ resistance first emerged in Southeast Asia early in the 1990s with a rapid increase from 0 to 84% over the period of 4 years [69] and has been widely spread to the other parts of the world, which might be due to an enhanced fitness of FQ-resistant isolates [70]. Significant portion of infections with FQ-resistant *Campylobacter* could be acquired through travel [71]. Extreme

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In Europe, FQ resistance in *Campylobacter* spp. was extremely high, but it varied among the sources of isolation, species and countries. In average, isolates of *C. coli* exhibited markedly higher resistance rates than isolates of *C. jejuni*. As seen in **Figure 6**, turkey (96.8% in *C. coli* and 76.2% in *C. jejuni*) and meat thereof (100% in *C. coli,* 74.5% in *C. jejuni*) presented the main

A rapid increase in FQ resistance in *Campylobacter* is evident in the last 14 years [27]. In Slovenia, for instance, the resistance level to nalidixic acid rapidly increased in isolates from broiler meat from 49.1% in 2001–2003 [73] to 78.6% in 2006 [11]. In 2016, in average, 77.3% of *Campylobacter* spp. from broilers exhibited FQ resistance [5]. Recent data suggest the presence and clonal spread of FQ-resistant *C. jejuni* clonal complex ST-21 in central Europe (Slovenia,

FQ resistance in *C. jejuni* (**Figure 7**) from broilers (in average 66.9%) varied from 8.4% in Finland to 97.9% in Latvia [5]. Furthermore, in 2014, Latvia reported on 100% resistant isolates

**Figure 6.** Sources and prevalence of FQ-resistant isolates of *C. jejuni* (turkey, turkey meat, broilers, broiler meat and human, respectively) and *C. coli* (turkey meat, turkeys, broilers, broiler meat, humans and pigs, respectively) [5, 22]*.*

**Figure 7.** Rates of FQ resistance in *C. jejuni* from broilers and humans, in *C. coli* from humans and combined resistance

**Figure 5.** Prevalence of resistance in *Campylobacter* spp*.* ERM: erythromycin, CIP: ciprofloxacin, TET: tetracyclines [5, 22]*.*

In general, resistance to erythromycin, the second clinically important antimicrobial for treating campylobacteriosis, is generally uncommon; however, more resistant isolates were detected in pigs (21.6% in 2015), as seen in **Figure 5** [22]*,* which could reflect a wide use of macrolides for the treatment of common infections in pigs [61]. In contrast to *C. jejuni* from humans (2.1%), markedly higher erythromycin resistance level was observed in *C. coli* (11.0%), with the highest proportion in Estonia (63.2%) and Portugal (50%). Similar trends could be observed in isolates from livestock and food [5]. Similarly, high levels of resistance (62.4%) were recorded in *C. coli* from pigs in Spain [22] and even higher in *Campylobacter* isolates from poultry meat in Italy (72.1%) [62].

Resistance to macrolides in *Campylobacter* most commonly occurs *via* chromosomal mutations in 23S rRNA [63] that reduces the binding affinity of macrolides to the binding site. These mutations were, however, demonstrated to have a fitness cost and to slow growth rates [64]. Recently, transferrable erythromycin resistance, conferred by the rRNA methylase *erm(B)* gene and located on either plasmids or associated with chromosomal multidrug resistance genomic islands, was detected in humans and livestock [65].

Southern Europe in average recorded higher prevalence of resistance to tetracyclines, which may also be used for the treatment of campylobacteriosis in humans, and is, in addition to FQ resistance, a very common feature [5]. Marked variations in tetracycline resistance could be observed between *C. coli* and *C. jejuni,* countries and sources of isolation. Resistance rates varied from very low (<10%) in *C. coli* from pigs in Sweden [66], moderate in *Campylobacter* spp. from cattle in Poland (20.9%) [52], *C. jejuni* from broiler carcasses in Belgium (47%) [67] and *C. jejuni* from chicken meat in France (53.6%) [51] to extremely high in isolates of *C. coli* from pigs in France (93%) [66], as well as *Campylobacter* spp. from quails in Portugal (96.7%) [68]. In general, *C. coli* exhibited higher resistance levels [5].

#### **4.3. FQ resistance and combined/MDR resistance in** *Campylobacter*

FQ resistance first emerged in Southeast Asia early in the 1990s with a rapid increase from 0 to 84% over the period of 4 years [69] and has been widely spread to the other parts of the world, which might be due to an enhanced fitness of FQ-resistant isolates [70]. Significant portion of infections with FQ-resistant *Campylobacter* could be acquired through travel [71]. Extreme rates of FQ resistance in endemic areas [72] are therefore of concern.

In Europe, FQ resistance in *Campylobacter* spp. was extremely high, but it varied among the sources of isolation, species and countries. In average, isolates of *C. coli* exhibited markedly higher resistance rates than isolates of *C. jejuni*. As seen in **Figure 6**, turkey (96.8% in *C. coli* and 76.2% in *C. jejuni*) and meat thereof (100% in *C. coli,* 74.5% in *C. jejuni*) presented the main sources of resistance, closely followed by broilers and meat thereof [5].

A rapid increase in FQ resistance in *Campylobacter* is evident in the last 14 years [27]. In Slovenia, for instance, the resistance level to nalidixic acid rapidly increased in isolates from broiler meat from 49.1% in 2001–2003 [73] to 78.6% in 2006 [11]. In 2016, in average, 77.3% of *Campylobacter* spp. from broilers exhibited FQ resistance [5]. Recent data suggest the presence and clonal spread of FQ-resistant *C. jejuni* clonal complex ST-21 in central Europe (Slovenia, Germany, Austria) [74].

FQ resistance in *C. jejuni* (**Figure 7**) from broilers (in average 66.9%) varied from 8.4% in Finland to 97.9% in Latvia [5]. Furthermore, in 2014, Latvia reported on 100% resistant isolates

In general, resistance to erythromycin, the second clinically important antimicrobial for treating campylobacteriosis, is generally uncommon; however, more resistant isolates were detected in pigs (21.6% in 2015), as seen in **Figure 5** [22]*,* which could reflect a wide use of macrolides for the treatment of common infections in pigs [61]. In contrast to *C. jejuni* from humans (2.1%), markedly higher erythromycin resistance level was observed in *C. coli* (11.0%), with the highest proportion in Estonia (63.2%) and Portugal (50%). Similar trends could be observed in isolates from livestock and food [5]. Similarly, high levels of resistance (62.4%) were recorded in *C. coli* from pigs in Spain [22] and even higher in *Campylobacter*

**Figure 5.** Prevalence of resistance in *Campylobacter* spp*.* ERM: erythromycin, CIP: ciprofloxacin, TET: tetracyclines [5, 22]*.*

Resistance to macrolides in *Campylobacter* most commonly occurs *via* chromosomal mutations in 23S rRNA [63] that reduces the binding affinity of macrolides to the binding site. These mutations were, however, demonstrated to have a fitness cost and to slow growth rates [64]. Recently, transferrable erythromycin resistance, conferred by the rRNA methylase *erm(B)* gene and located on either plasmids or associated with chromosomal multidrug resistance

Southern Europe in average recorded higher prevalence of resistance to tetracyclines, which may also be used for the treatment of campylobacteriosis in humans, and is, in addition to FQ resistance, a very common feature [5]. Marked variations in tetracycline resistance could be observed between *C. coli* and *C. jejuni,* countries and sources of isolation. Resistance rates varied from very low (<10%) in *C. coli* from pigs in Sweden [66], moderate in *Campylobacter* spp. from cattle in Poland (20.9%) [52], *C. jejuni* from broiler carcasses in Belgium (47%) [67] and *C. jejuni* from chicken meat in France (53.6%) [51] to extremely high in isolates of *C. coli* from pigs in France (93%) [66], as well as *Campylobacter* spp. from quails in Portugal (96.7%)

isolates from poultry meat in Italy (72.1%) [62].

20 Antimicrobial Resistance - A Global Threat

genomic islands, was detected in humans and livestock [65].

[68]. In general, *C. coli* exhibited higher resistance levels [5].

**Figure 6.** Sources and prevalence of FQ-resistant isolates of *C. jejuni* (turkey, turkey meat, broilers, broiler meat and human, respectively) and *C. coli* (turkey meat, turkeys, broilers, broiler meat, humans and pigs, respectively) [5, 22]*.*

**Figure 7.** Rates of FQ resistance in *C. jejuni* from broilers and humans, in *C. coli* from humans and combined resistance to FQ and erythromycin in *C. coli* from humans [5].

of *Campylobacter* from chicken [75]. In humans, the highest rates of FQ resistance were reported for *C. coli* from Italy and Portugal (100%) and in for *C. jejuni* from Portugal and Estonia (>90%). Notably, 9 out of 19 EU MS recorded 80–100% resistance rates for *C. coli* (**Figure 7**) [5]*.*

In recent years, the development of high-throughput technologies and platforms for massive DNA sequencing, and genomics tools has opened new possibilities also in the surveillance of AMR in common zoonotic bacteria. WGS, together with appropriate databases, general (NCBI, ENA) or specialised for AMR (ARG-ANNOT, ResFinder, CARD, RED-DB, Bacmet), bioinformatic tools (BLAST) and platforms enable detection of antibiotic resistance genetic loci in the genomes of bacterial isolates or microbiomes and reveal the mechanisms leading to AMR. While WGS offers very rapid and efficient tool for detection of the antibiotic resistance genes (ARG) in genomes of individual bacterial isolates, the main issue remains how to predict from these data the actual antimicrobial susceptibility, and epidemiological or clinical cut-off values [85]. However, differentiation among isolates with acquired or intrinsic resistance on the basis of phenotypic MIC determinations only is also not totally accurate. Furthermore, it should be considered that also the strains that contain the genes associated with antimicrobial resistance but do not exhibit phenotypic resistance present certain risk for the horizontal spread when consumed.

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The usefulness of WGS for antimicrobial resistance surveillance was confirmed in several studies. Examination of 640 nontyphoidal *Salmonella* isolates from retail meat and human clinical samples identified known resistance genes and phenotypic resistance to 14 antimicrobials, where the correlation between resistance genotypes and phenotypes was close to 100% for most classes of antibiotics, and lower for aminoglycosides and beta-lactams [86]**.** In addition to known ARG, several unique resistance genes were found, more in the human isolates (n = 59) than in the retail meat isolates (n = 36). The authors concluded that the use of more appropriate MIC breakpoints and inclusion of new AGSs in the databases will further improve the correlations between phenotypic and genotypic observations. For *Salmonella typhimurium* isolates (n = 50) from Danish pigs, high concordance (99.74%) between phenotypic and predicted antimicrobial susceptibility was observed as well [87]. Phenotypic resistance to quinolones and fluoroquinolones due to chromosomal mutations, however, could

Genomic approach is increasingly used also in the developing of control methods and identification of antimicrobial resistance markers for evidence-based decisions in epidemiology and surveillance of foodborne diseases. OMICS datasets have been found as a powerful tool to complement current studies that are starting to be used also in some risk assessment areas. In a current comprehensive study "Syst-OMICS," 4500 *Salmonella* genomes will be sequenced and analysis pipeline built in order to study *Salmonella* genome evolution, antibiotic resistance and virulence genes [88]. The data of the first 3377 genomes already sequenced are stored in the newly established *Salmonella* Foodborne Syst-OMICS database (SalFoS, https://salfos.ibis. ulaval.ca/). Their analysis identified 1003 unique resistomes, composed of combinations of 195 different genes. Surprisingly, the two most frequently observed resistomes accounted for

Comparative genomics of the WGS was successfully used also in the examination of 589 *Campylobacter* isolates from retail chicken meat exhibiting phenotypic resistance to 9 antimicrobials [89]. For most antimicrobial agents (ciprofloxacin, nalidixic acid, gentamicin, azithromycin, erythromycin and clindamycin), the observed phenotypic resistance, determined on the basis

not be detected by ResFinder platform.

23% of the *Salmonella* strains examined.

Overall, 9.2% of human *C. coli* exhibited combined resistance to ciprofloxacin, erythromycin and tetracycline with resistance rates ranging from 0 to 57.9% (Estonia), which is shown in **Figure 7** [5]*.* Erythromycin resistance is often associated with MDR phenotype [63]. In Finland, for example, 94.7% of *Campylobacter* isolates from humans were, in addition to erythromycin, resistant to FQ, and 73.7% to tetracycline [76]. Combined resistance to the first-line drugs may be associated with adverse events such as delayed recovery, invasive illness and prolonged treatment with feasible alternative antimicrobials [77, 78].

FQ resistance in *C. jejuni* and *C. coli* can be mediated through specific point mutations in *gyrA* gene, encoding for DNA gyrase or through chromosomally encoded multidrug efflux pump. The two mechanisms work synergistically [79]. Efflux pumps in *Campylobacter,* primarily CmeABC, are involved in resistance to broad spectrum of antimicrobials, including macrolides and quinolones [80], as well as cross-resistance to other compounds such as bile salts [81]. Therapeutic application of efflux pump inhibitors (e.g., epigallocatechin gallate) that were shown to restore macrolide efficacy could be a feasible treatment option in combination with the macrolide therapy [80, 82–84].
